To avoid photodamage, photosynthetic organisms are able to thermally dissipate the energy absorbed in excess in a process known as nonphotochemical quenching (NPQ). Although NPQ has been studied extensively, the major players and the mechanism of quenching remain debated. This is a result of the difficulty in extracting molecular information from in vivo experiments and the absence of a validation system for in vitro experiments. Here, we have created a minimal cell of the green alga Chlamydomonas reinhardtii that is able to undergo NPQ. We show that LHCII, the main light harvesting complex of algae, cannot switch to a quenched conformation in response to pH changes by itself. Instead, a small amount of the protein LHCSR1 (light-harvesting complex stress related 1) is able to induce a large, fast, and reversible pH-dependent quenching in an LHCII-containing membrane. These results strongly suggest that LHCSR1 acts as pH sensor and that it modulates the excited state lifetimes of a large array of LHCII, also explaining the NPQ observed in the LHCSR3-less mutant. The possible quenching mechanisms are discussed.photosynthesis | light-harvesting | nonphotochemical quenching | green algae | thylakoid membranes P hotosynthetic organisms get their energy from light and have developed a series of mechanisms to respond to the changes in light intensity that occur in their natural environment (1, 2). This is particularly important in high-light conditions, as the energy absorbed in excess can induce photodamage, eventually leading to the death of the organism. Photosynthetic organisms are equipped with many pigment-protein complexes, most of which in plants and green algae are members of the light-harvesting complex (Lhc) multigenic family (3). These complexes maximize light absorption in low light, but can easily become overexcited in high light (4), when a large part of the absorbed light cannot be used for charge separation in the reaction centers of the photosystems. Especially when the changes in light intensity are very fast, and thus protein degradation is not an option, photoprotective mechanisms need to be switched on to avoid the formation of singlet oxygen. The most rapid response to high light intensity is the dissipation of a large part of the absorbed energy as heat in a series of processes known as nonphotochemical quenching (NPQ) (1,5,6).The general idea is that the LHCs can switch between a lightharvesting conformation, characterized by a long excited-state lifetime, and a quenched (Q) conformation that shows a shorter lifetime because of the presence of competing de-excitation processes (7). How this switch is induced and the nature of these de-excitation processes is a matter of debate. It is known that NPQ is triggered by low luminal pH, which is a signal for the overexcitation of the membrane; this activates the quenching processes (5, 8), which involves the proteins PsbS (in plants and mosses) (9, 10) and/or lightharvesting complex stress related (LHCSR) (in green algae, mosses, and diatoms) (10-12)....

Photosynthetic organisms are exposed to drastic changes in light conditions, which can affect their photosynthetic efficiency and induce photodamage. To face these changes, they have developed a series of acclimation mechanisms. In this work, we have studied the acclimation strategies of Chlamydomonas reinhardtii, a model green alga that can grow using various carbon sources and is thus an excellent system in which to study photosynthesis. Like other photosynthetic algae, it has evolved inducible mechanisms to adapt to conditions where carbon supply is limiting. We have analyzed how the carbon availability influences the composition and organization of the photosynthetic apparatus and the capacity of the cells to acclimate to different light conditions. Using electron microscopy, biochemical, and fluorescence measurements, we show that differences in CO 2 availability not only have a strong effect on the induction of the carbon-concentrating mechanisms but also change the acclimation strategy of the cells to light. For example, while cells in limiting CO 2 maintain a large antenna even in high light and switch on energy-dissipative mechanisms, cells in high CO 2 reduce the amount of pigments per cell and the antenna size. Our results show the high plasticity of the photosynthetic apparatus of C. reinhardtii. This alga is able to use various photoacclimation strategies, and the choice of which to activate strongly depends on the carbon availability.

In photosynthetic eukaryotes, thousands of proteins are translated in the cytosol and imported into the chloroplast through the concerted action of two translocons—termed TOC and TIC—located in the outer and inner membranes of the chloroplast envelope, respectively. The degree to which the molecular composition of the TOC and TIC complexes is conserved over phylogenetic distances has remained controversial. Here, we combine transcriptomic, biochemical, and genetic tools in the green alga Chlamydomonas (Chlamydomonas reinhardtii) to demonstrate that, despite a lack of evident sequence conservation for some of its components, the algal TIC complex mirrors the molecular composition of a TIC complex fromArabidopsis thaliana.The Chlamydomonas TIC complex contains three nuclear-encoded subunits, Tic20, Tic56, and Tic100, and one chloroplast-encoded subunit, Tic214, and interacts with the TOC complex, as well as with several uncharacterized proteins to form a stable supercomplex (TIC-TOC), indicating that protein import across both envelope membranes is mechanistically coupled. Expression of the nuclear and chloroplast genes encoding both known and uncharacterized TIC-TOC components is highly coordinated, suggesting that a mechanism for regulating its biogenesis across compartmental boundaries must exist. Conditional repression of Tic214, the only chloroplast-encoded subunit in the TIC-TOC complex, impairs the import of chloroplast proteins with essential roles in chloroplast ribosome biogenesis and protein folding and induces a pleiotropic stress response, including several proteins involved in the chloroplast unfolded protein response. These findings underscore the functional importance of the TIC-TOC supercomplex in maintaining chloroplast proteostasis.

LHCII is the major antenna complex of plants and algae, where it is involved in light harvesting and photoprotection. Its properties have been extensively studied in vitro, after isolation of the pigment-protein complex from the membranes, but are these properties representative for LHCII in the thylakoid membrane? In this work, we have studied LHCII in the cells of the green alga C. reinhardtii acclimated to different light conditions in the absence of the other components of the photosynthetic apparatus. We show that LHCII exists in the membranes in different fluorescence quenching states, all having a shorter excited-state lifetime than isolated LHCII in detergent. The ratio between these populations depends on the light conditions, indicating that the light is able to regulate the properties of the complexes in the membrane.

The efficient use of excitation energy in photosynthetic membranes is achieved by a dense network of pigment-protein complexes. These complexes fulfill specific functions and interact dynamically with each other in response to rapidly changing environmental conditions. Here, we studied how in the intact cells of Chlamydomonas reinhardtii (C.r.) the lack of the photosystem I (PSI) core or the photosystem II (PSII) core affects these interactions. To that end the mutants F15 and M18 (both PSI-deficient) and FUD7 (PSII-deficient) were incubated under conditions known to promote state transitions in wild-type. The intact cells were then instantly frozen to 77K and the full-spectrum time-resolved fluorescence emission of the cells was measured by means of streak camera. In the PSI-deficient mutants excitation energy transfer (EET) towards light-harvesting complexes of PSI (Lhca) occurs in less than 0.5 ns, and fluorescence from Lhca decays in 3.1 ns. Decreased trapping by PSII and increased fluorescence of Lhca upon state 1 (S1)→state 2 (S2) transition appears in the F15 and less in the M18 mutant. In the PSII-deficient mutant FUD7, quenched (0.5 ns) and unquenched (2 ns) light-harvesting complexes of PSII (LHCII) are present in both states, with the quenched form more abundant in S2 than in S1. Moreover, EET of 0.4 ns from the remaining LHCII to PSI increases upon S1→S2 transition. We relate the excitation energy kinetics observed in F15, M18 and FUD7 to the remodeling of the photosynthetic apparatus in these mutants under S1 and S2 conditions.